Dimeric compounds and their use as anti-viral agents

ABSTRACT

The invention relates to compounds of general formula (I); in which R is an amino or guanidino group; R 2  is acetyl or trifluoroacetyl; X is CONH, NHCO or O; n is an integer from 2 to 6; and Y is C 2 –C 8  alkyl C 3-8  cycloalkyl, C 1 –C 4  alkoxyalkyl, an amino acid or dipeptide, or a pharmaceutically acceptable derivative thereof, methods for their preparation, pharmaceutical formulations containing them or their use in the prevention or treatment of a viral infection

This invention relates to new chemical compounds and their use inmedicine. In particular the invention concerns novel dimeric compounds,methods for their preparation, pharmaceutical formulations thereof andtheir use as anti-viral agents.

BACKGROUND OF THE INVENTION

Enzymes with the ability to cleave N-acetyl neuraminic acid (NANA), alsoknown as sialic acid, from other carbohydrates are present in manymicroorganisms. These include bacteria such as Vibrio cholerae,Clostridium perfringens, Streptococcus pneumoniae and Arthrobactersialophilus, and viruses such as influenza virus, parainfluenza virus,mumps virus, Newcastle disease virus and Sendai virus. Most of theseviruses are of the orthomyxovirus or paramyxovirus groups, and carry aneuraminidase activity on the surface of the virus particles. Many ofthese neuraminidase-possessing organisms are major pathogens of manand/or animals, and some, such as influenza virus and Newcastle diseasevirus, cause diseases of enormous importance.

It has long been thought that inhibitors of neuraminidase might preventinfection by neuraminidase-bearing viruses. Most of the knownneuraminidase inhibitors are analogues of neuraminic acid, such as2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) and some of itsderivatives (Meindl et al, Virology, 1974 58 457). Our InternationalPatent Publication No. WO 91/16320 describes a number of analogues ofDANA which are active against viral neuraminidase, and it has been shownin particular that 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminicacid (Compound (A), code number GG167) is useful in the treatment ofinfluenza A and B (N. Engl. J. Med., 1997 337 874–880). Other patentapplications describe various closely-related sialic acid derivatives(eg. PCT Publications No. WO 95/18800, No. WO 95/20583 and No. WO98/06712), and anti-viral macromolecular conjugates of GG167 have alsobeen described (International Patent Application No. PCT/AU97/00771).

Ac represents acetyl

International Patent Publication No. WO 00/55149, describes dimericcompounds which comprise two neuraminidase binding molecules, such ascompound (A), attached to a common spacer or linking group of up to 100atoms in length.

We have now discovered a novel class of compounds which fall within thegeneric scope of International Patent Publication No. WO 00/55149, butwhich are not specifically disclosed therein, and exhibit a surprisinglyadvantageous anti-influenza activity profile which includes a long lungresidency time and high potency.

Without wishing to be bound by theory, the basis for the long residencytime in the lungs is thought to be due to the size and molecular weightof the compounds preventing entry through tight junctions in therespiratory epithelium and the polarity of the compounds being such thatpassage through the cell membranes occurs very inefficiently. Analternative theory is that the compounds themselves interact with thephospholipids in the cell membrane or other components of therespiratory epithelium and increase the residency time in the lungs.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides for a compound ofgeneral formula (I):

in which

R is an amino or guanidino group;

R² is acetyl or trifluoroacetyl;

X is CONH, NHCO or O;

n is an integer from 2 to 6; and

Y is C₂–C₈ alkyl C₃₋₈ cycloalkyl, C₁–C₄ alkoxyalkyl, an amino acid ordipeptide,

or a pharmaceutically acceptable derivative thereof, with the provisothat when R is a guanidino group, R² is acetyl, X is O and n is 2, thenY is not C₂ alkyl.

Preferably R is a guanidino group.

Preferably R² is an acetyl group.

The term “C₂–C₈ alkyl” refers to straight chain or branched chainhydrocarbon groups having from 2 to 8 carbon atoms. Illustrative of suchalkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, heptyl and octyl.

As used herein, the term “C₃–C₈ cycloalkyl” refers to an alicyclic grouphaving from 3 to 8 carbon atoms. Illustrative of such cycloalkyl groupsare cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

As used herein the term “C₁–C₄ alkoxyalkyl” refers to straight chain orbranched chain alkoxy groups having from 1 to 4 carbon atoms.Illustrative of such alkoxy groups are methoxy, ethoxy, propoxy,isopropoxy, butoxyl, isobutoxy, sec-butoxy and tert-butoxy.

The term “amino acid” is used herein in its broadest sense and refers toα-amino substituted carboxylic acids which are the building blocks ofproteins such as the 20 standard amino acids as listed at page 96 of“Principles of Biochemistry” by Albert L. Lehninger (Worth Publishers,Inc. 1982).

The term “dipeptide” is used herein in its broadest sense and refers totwo amino acids linked by a peptide bond (also called an amide bond).Examples include glutamic-glycine, glycine-glycine or aspartic-glycine.

It will be appreciated by those skilled in the art that the compounds offormula (I) may be modified to provide pharmaceutically acceptablederivatives thereof at any one or more of the functional groups in thecompounds of formula (I). Of particular interest as such derivatives arecompounds modified at the carboxyl function, hydroxyl functions or atamino groups. Thus compounds of interest include alkyl esters, such asmethyl, ethyl, propyl or isopropyl esters, aryl esters, such as phenyl,benzoyl esters, and acetyl esters of the compounds of formula (I).

The term “pharmaceutically acceptable derivative” means anypharmaceutically acceptable salt, ether, ester or salt of such ester ofa compound of formula (I) or any other compound which, uponadministration to the recipient, is capable of providing a compound offormula (I) or an anti-virally active metabolite or residue thereof. Ofparticular interest as derivatives are compounds modified at the sialicacid carboxy or glycerol hydroxy groups, or at amino and guanidinegroups.

Pharmaceutically acceptable salts of the compounds of formula (I)include those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acids includehydrochloric, hydrobromic, sulphuric, nitric, perchloric, fumaric,maleic, phosphoric, glycollic, lactic, salicylic, succinic,toluene-p-sulphonic, tartaric, acetic, citric, methanesulphonic, formic,benzoic, malonic, naphthalene-2-sulphonic and benzenesulphonic acids.Other acids such as oxalic acid, while not in themselvespharmaceutically acceptable, may be useful in the preparation of saltsuseful as intermediates in obtaining compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

Salts derived from appropriate bases include alkali metal (eg. sodium),alkaline earth metal (eg. magnesium), ammonium, and NR₄ ⁺ (where R isC₁₋₄alkyl) salts.

The compounds of the invention may be prepared by methods describedherein. It will be apparent to those skilled in the art, that it isnecessary to use protecting groups to protect one or more functionalgroups of the neuraminidase binding molecule during the process ofattaching the monomers to the alkyl spacer group. See for example“Protective Groups in Organic Synthesis” by T. W. Green and P. G. M.Nuts (John Wiley & Sons, 1991). Pharmaceutically acceptable salts of thecompounds of formula (I) may be prepared according to known procedures.

For ease of processing and preparation, it is preferable that thecompounds of formula (I) are in crystalline form.

Accordingly, the present invention also provides a method for thepreparation of the compound of formula (I) as defined above when X isCONH or NHCO, which comprises the steps of

(a) coupling a compound of formula (II)

in which R² and n are as defined above, P₁ is a carboxylic acidprotecting group and X₁ is NH₂ or CO₂H;

with a compound of formula (III)X₂—Y—X₂  (III)

in which Y is as defined above and X₂ is CO₂H or NH₂ provided that it isnot the same as X₁ to form a compound of formula (IV)

-   -   (b) deprotecting the compound of formula (IV).

The compounds of formula (I) possess antiviral activity. In particularthese compounds are inhibitors of viral neuraminidase oforthomyxoviruses and paramyxoviruses, for example the viralneuraminidase of influenza A and B, parainfluenza, mumps and Newcastledisease.

Thus in a second aspect the invention provides a compound of formula (I)or a pharmaceutically acceptable derivative thereof, for use as anactive therapeutic agent in the treatment of a viral infection, forexample orthomyxovirus and paramyxovirus infections.

In a third aspect the invention provides a method for the prevention ortreatment of a viral infection comprising the step of administration toa subject in need thereof of an effective amount of a compound offormula (I), or a pharmaceutically acceptable salt or derivativethereof.

Preferably, the viral infection is an orthomyxovirus or paramyxovirusinfection. More preferably the viral infection is an influenza A or Binfection.

Preferably the subject is an animal such as a mammal, more preferably ahuman, or a member of the genus Equus, for example a horse, donkey ormule. Most preferably the mammal is a human.

In a fourth aspect the invention provides use of a compound of theinvention for the manufacture of a medicament for the treatment of aviral infection.

As used herein, the term “effective amount” is meant an amount of thecompound of formula I effective to preventing or treating a viralinfection in order to yield a desired therapeutic response. For example,to overcome or alleviate the effects of a viral infection.

The term “therapeutically-effective amount” means an amount of thecompound of formula I to yield a desired therapeutic response. Forexample, treating or preventing a viral infection.

The specific “therapeutically-effective amount” will, obviously, varywith such factors as the particular viral infection being treated, thephysical condition of the subject, the type of animal being treated, theduration of the treatment, the nature of concurrent therapy (if any),and the specific formulation employed and the structure of the compoundor its derivatives.

Generally, the terms “treating”, “treatment” and the like are usedherein to mean affecting a subject, tissue or cell to obtain a desiredpharmacologic and/or physiologic effect. The effect may be prophylacticin terms of completely or partially preventing a viral infection or signor symptom thereof, and/or may be therapeutic in terms of a partial orcomplete cure of a viral infection. “Treating” as used herein covers anytreatment of, or prevention of a viral infection in a vertebrate, amammal, particularly a human, and includes: (a) preventing the viralinfection from occurring in a subject that may be predisposed to theviral infection, but has not yet been diagnosed to the viral infection,but has not yet been diagnosed as having it; (b) inhibiting the viralinfection, ie., arresting its development; or (c) relieving orameliorating the effects, i.e., cause regression of the symptoms of theviral infection.

The compounds of the invention may also be used in diagnostic methods,in particular methods for the detection of influenza virus. For use insuch methods it may be advantageous to link a compound of the inventionto a label, such as a radioactive, fluorescent or chemiluminescentlabel.

Methods of diagnosis for which the compounds of the invention aresuitable are described, for example, in our earlier applicationsPCT/AU97/00109 and PCT/AU97/00771.

In a fifth aspect the invention provides a method for the detection of aviral infection which comprises the step of contacting the compound ofthe invention with a sample suspected of containing the virus.

It will be further appreciated that the amount of a compound of theinvention required for use in treatment will vary not only with theparticular compound selected but also with the route of administration,the nature of the condition being treated, and the age and condition ofthe patient, and will ultimately be at the discretion of the attendantphysician or veterinarian. In general however, a suitable dose will bein the range of from about 0.001 to 100 mg/kg of bodyweight per day,preferably in the range of 0.01 to 10 mg/kg/day, most preferably in therange of 0.1 to 1 mg/kg/day.

Treatment is preferably commenced before or at the time of infection andcontinued until virus is no longer present in the respiratory tract.However the compounds are also effective when given post-infection, forexample after the appearance of established symptoms.

Suitably treatment is given on one or two occasions, preferably onlyonce only for treatment, and preferably once per week for prophylaxis.

The compound is conveniently administered in unit dosage form, forexample containing 1 to 100 mg, more conveniently 1 to 20 mg of activeingredient per unit dosage form.

While it is possible that, for use in therapy, a compound of theinvention may be administered as the raw chemical, it is preferable topresent the active ingredient as a pharmaceutical formulation.

Thus in a sixth aspect the invention provides a pharmaceuticalformulation comprising a compound of formula (I) or a pharmaceuticallyacceptable salt or derivative thereof, together with one or morepharmaceutically acceptable carriers therefor and, optionally, othertherapeutic and/or prophylactic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not being deleterious to the recipient thereof.

The compounds of the invention may also be used in combination withother therapeutic and/or prophylactic agents, for example otheranti-infective agents. In particular the compounds of the invention maybe employed with other antiviral agents. The invention thus provides ina seventh aspect a combination comprising a compound of formula (I) or apharmaceutically acceptable salt or derivative thereof together withanother therapeutically and/or prophylactically active agent, inparticular an antiviral agent.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus such formulationscomprising a combination as defined above together with apharmaceutically acceptable carrier therefore comprise a further aspectof the invention.

Suitable therapeutic and/or prophylactic agents for use in suchcombinations include other anti-infective agents, in particularanti-bacterial and anti-viral agents such as those used to treatrespiratory infections. For example, other compounds or vaccineseffective against influenza viruses, such as the sialic acid analoguesreferred to above, e.g. zanamivir, oseltamivir, amantadine, rimantadineand ribavirin and FluVax, may be included in such combinations.

The individual components of such combinations may be administeredeither separately, sequentially or simultaneously in separate orcombined pharmaceutical formulations.

When the compounds of the invention are used with a second therapeuticand/or prophylactic agent active against the same virus, the dose ofeach compound may either be the same as or different from that employedwhen each compound is used alone. Appropriate doses will be readilyappreciated by those skilled in the art.

Pharmaceutical formulations include those suitable for oral, rectal,nasal, topical (including buccal and sub-lingual), vaginal or parenteral(including intramuscular, sub-cutaneous and intravenous) administration,or those in a form suitable for administration to the respiratory tract(including the nasal passages) for example by inhalation orinsufflation. The formulations may, where appropriate, be convenientlypresented in discrete dosage units, and may be prepared by any of themethods well known in the art of pharmacy. These methods include thestep of bringing into association the active compound with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration mayconveniently be presented as discrete units such as capsules, cachets ortablets each containing a predetermined amount of the active ingredient;as a powder or granules; as a solution, a suspension or as an emulsion.The active ingredient may also be presented as a bolus, electuary orpaste. Tablets and capsules for oral administration may containconventional excipients such as binding agents, fillers, lubricants,disintegrants, or wetting agents. The tablets may be coated according tomethods well known in the art. Oral liquid preparations may for examplebe in the form of aqueous or oily suspensions, solutions, emulsions,syrups or elixirs, or may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles, which may includeedible oils, or preservatives.

The compounds according to the invention may also be formulated forparenteral administration by injection, for example bolus injection, orcontinuous infusion, and may be presented in unit dose form in ampoules,pre-filled syringes, small volume infusion or in multi-dose containerswith an added preservative. The compositions may take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, andmay contain formulating agents such as suspending, stabilising and/ordispersing agents. Alternatively, the active ingredient may be in powderform, obtained by aseptic isolation of sterile solid or bylyophilisation from solution, for constitution with a suitable vehicle,eg. sterile, pyrogen-free water, before use.

For topical administration to the epidermis the compounds according tothe invention may be formulated as ointments, creams or lotions, or as atransdermal patch. Ointments and creams may, for example, be formulatedwith an aqueous or oily base with the addition of suitable thickeningand/or gelling agents. Lotions may be formulated with an aqueous or oilybase, and will in general also contain one or more emulsifying agents,stabilising agents, dispersing agents, suspending agents, thickeningagents, or colouring agents.

Formulations suitable for topical administration in the mouth includelozenges comprising active ingredient in a flavoured base, usuallysucrose and gum acacia or gum tragacanth; pastilles comprising theactive ingredient in an inert base such as gelatin or sucrose and gumacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the active compound with thesoftened or melted carrier(s) followed by chilling and shaping moulds.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

For administration to the respiratory tract, including intranasaladministration, the neuraminidase inhibitors may be administered by anyof the methods and formulations employed in the art for administrationto the respiratory tract.

Thus in general the compounds may be administered in the form of asolution or a suspension or as a dry powder.

Solutions and suspensions will generally be aqueous, for exampleprepared from water alone (for example sterile or pyrogen-free water) orwater and a physiologically acceptable co-solvent (for example ethanol,propylene glycol or polyethylene glycols such as PEG 400).

Such solutions or suspensions may additionally contain other excipientsfor example preservatives (such as benzalkonium chloride), solubilisingagents/surfactants such as polysorbates (eg. Tween 80, Span 80,benzalkonium chloride), buffering agents, isotonicity-adjusting agents(for example sodium chloride), absorption enhancers and viscosityenhancers. Suspensions may additionally contain suspending agents (forexample microcrystalline cellulose, carboxymethyl cellulose sodium).

Solutions or suspensions are applied directly to the nasal cavity byconventional means, for example with a dropper, pipette or spray. Theformulations may be provided in single or multidose form. In the lattercase a means of dose metering is desirably provided. In the case of adropper or pipette this may be achieved by the patient administering anappropriate, predetermined volume of the solution or suspension. In thecase of a spray this may be achieved for example by means of a meteringatomising spray pump.

Administration to the respiratory tract may also be achieved by means ofan aerosol formulation in which the compound is provided in apressurised pack with a suitable propellant, such as achlorofluorocarbon (CFC), for example dichlorodifluoromethane,trichlorofluoromethane or dichlorotetrafluoroethane, carbon dioxide orother suitable gas. The aerosol may conveniently also contain asurfactant such as lecithin. The dose of drug may be controlled byprovision of a metered valve.

Alternatively the compounds may be provided in the form of a dry powder,for example a powder mix of the compound in a suitable powder base suchas lactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidine (PVP). Conveniently the powdercarrier will form a gel in the nasal cavity. The powder composition maybe presented in unit dose form, for example in capsules or cartridges ofeg. gelatin, or blister packs from which the powder may be administeredby means of an inhaler.

In formulations intended for administration to the respiratory tract,including intranasal formulations, the compound will generally have asmall particle size, for example of the order of 5 microns or less. Sucha particle size may be obtained by means known in the art, for exampleby micronisation.

When desired, formulations adapted to give sustained release of theactive ingredient may be employed.

Preferably the compounds of the invention are administered to therespiratory tract by inhalation, insufflation or intranasaladministration, or a combination thereof.

“Relenza” is administered by oral inhalation as a free-flow powder via a“Diskhaler” (trade marks of the GlaxoSmithKline group of companies). Asimilar formulation would be suitable for the present invention.

Thus, according to an eighth aspect of the present invention there isprovided an inhaler which contains a formulation as defined above.

It will be appreciated that the inhaler may also be in the form of ameter dose aerosol inhaler.

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail by way of reference onlyto the following non-limiting examples.

TABLE 1

in which R² is acetyl and R is guanidine Compound No. Y X n 1 (CH₂)₈CONH 6 2 (CH₂)₄ CONH 6 3 (CH₂)₂ CONH 6 4 CH₂OCH₂ CONH 6 5 (CH₂)₃ CONH 66 (CH₂)₅ CONH 6 7 (CH₂)₆ CONH 6 8 (CH₂)₇ CONH 6 9 (CH₂)₂ CONH 3 105-Norbornene-2,3-diacid CONH 6 11 (CH₂)₂ CONH 3 12 CH₂CH₂CH(NH₂)CONHCH₂CONH 3 (Glutamic-glycine) 13 CH(NH₂)CH₂CH₂ CONH 3 (Glutamic) 14CH(NH₂)CH₂ CONH 3 (Aspartic) 15 (CH₂)₃ NHCO 2 16 CH₂CH₂OCH₂CH₂ NHCO 2 17(CH₂)₂ NHCO 2 18 (CH₂)₆ NHCO 2 19 (CH₂)₄ NHCO 1 20 (CH₂)₆ NHCO 1 21(CH₂)₄ CONH 3 22 (CH₂)₅ CONH 3 23 (CH₂)₆ CONH 3 24 (CH₂)₇ CONH 3 25(CH₂)₈ CONH 3Machine MethodsGreen Method (LC/MS)

Micromass Platform II mass spectrometer operating in positive ionelectrospray mode, mass range 100–1000 amu.

-   Column: 3.3 cm×4.6 mm ID, 3 μm ABZ+PLUS-   Flow Rate: 3 ml/min-   Injection Volume: 5 μl-   Solvent A: 95% acetonitrile+0.05% formic acid-   Solvent B: 0.1% formic acid+10 mMolar ammonium acetate-   Gradient: 0% A/0.7 min, 0–100% A/3.5 min, 100% A/1.1 min, 100–0%    A/0.2 min    Purple Method (Mass Directed Autoprep HPLC)

The prep column used was a Supelcosil ABZplus (10 cm×2.12 cm)

-   UV wavelength: 200–320 nM-   Flow: 20 ml/min-   Injection Volume: 1 ml-   Solvent A: 0.1% formic acid-   Solvent B: 95% acetonitrile+5% formic acid-   Gradient: 100% A/1 min, 100–80% A/9 min, 80–1% A/3.5 min, 1% A/1.4    min, 1–100% A/0.1 min    Turquoise Method (Autoprep HPLC)

The prep column used was a Supelcosil ABZplus (10 cm×2.12 cm).

-   UV wavelength: 230 nm-   Flow: 4 ml/min-   Injection Volume: 2 ml-   Solvent A: acetonitrile+0.05% TFA-   Solvent B: water+0.1% TFA    Method A(LC/MS)

Micromass Platform II mass spectrometer operating in positive ionelectrospray mode, mass range 100–1000 amu.

-   Column: 3.3 cm×4.6 mm ID, 3 μm ABZ+PLUS-   Flow Rate: 3 ml/min-   Injection Volume: 5 μl-   Solvent A: 95% acetonitrile+0.05% formic acid-   Solvent B: 0.1% formic acid+10 mMolar ammonium acetate-   Gradient: 0% A/0.7 min, 0–100% A/3.5 min, 100% A/1.1 min, 100–0%    A/0.2 min    Method B (LC/MS)

Waters ZQ mass spectrometer operating in positive ion electrospray mode,mass range 100–1000 amu.

-   Column: 3.3 cm×4.6 mm ID, 3 μm ABZ+PLUS-   Flow Rate: 3 ml/min-   Injection Volume: 5 μl-   Solvent A: 95% acetonitrile+0.05% formic acid-   Solvent B: 0.1% formic acid+10 mMolar ammonium acetate-   Gradient: 0% A/0.7 min, 0–100% A/3.5 min, 100% A/1.1 min, 100–0%    A/0.2 min    Method C (Autoprep HPLC)

The prep column used was a Supelcosil ABZplus (10 cm×2.12 cm).

-   UV wavelength: 230 nm-   Flow: 4 ml/min-   Injection Volume: 2 ml-   Solvent A: acetonitrile+0.05% TFA-   Solvent B: water+0.1% TFA-   Gradient: 0–40% A/20 min, 40% A/20 min, 40–100% A/0.3 min, 100% A/15    min, 100–0% A/3 min    Method D (Mass Directed Autoprep HPLC)

The prep column used was a Supelcosil ABZplus (10 cm×2.12 cm)

-   UV wavelength: 200–320 nM-   Flow: 20 ml/min-   Injection Volume: 1 ml-   Solvent A: 0.1% formic acid-   Solvent B: 95% acetonitrile+5% formic acid-   Gradient: 100% A/1 min, 100–80% A/9 min, 80–1% A/3.5 min, 1% A/1.4    min, 1–100% A/0.1 min    Abbreviations-   EtOAc ethyl acetate-   MeOH methanol-   HPLC high pressure liquid chromatography-   SPE solid phase extraction-   LC/MS Liquid chromatography/mass spectroscopy-   DMF N,N-Dimethylformamide-   WSCDI 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide methiodide-   HOBt 1-hydroxybenzotriazole-   DIPEA N,N-diisopropylethylamine-   MeCN acetonitrile-   RT room temperature-   EtOAc ethyl acetate-   MgSO₄ magnesium sulphate-   DMF dimethylformamide

(4S, 5R,6R)-5-Acetylamino-4-azido-6-[(S)-4-nitrophenoxycarbonyloxy)-(2-oxo-[1,3]dioxolan-4R-yl)-methyl]-5,6-dihydro-4H-pyran-2-carboxylicacid methylester (see Eur. J. Med. Chem. 1999, 34, 563–574) (2.00 g, 3.8mmol) was dried by azeotroping 3 times from anhydrous toluene and thendissolved in anhydrous acetonitrile (20 ml) with the addition of a few 3angstrom molecular sieve pellets. The stirred solution was treated withN-tert-butoxycarbonyl 1,4-diaminobutane (0.72 g, 3.8 mmol) andtriethylamine (0.43 g, 4.2 mmol). The mixture was stirred for 16 h undera nitrogen atmosphere. Volatiles were removed in vacuo to afford ayellow residue. This was redissolved in EtOAc (50 ml), washed with 0.5MHCl (30 ml) then brine (30 ml). The solution was dried (Na₂SO₄) andsolvent evaporated in vacuo to afford a cream coloured foam. Furtherpurification was by Biotage flash chromatography, eluant initiallyEtOAc: Cyclohexane (1:1) then EtOAc. Evaporation of solvent in vacuogave Intermediate 1 (1.26 g, 58% yield) as a white solid. LC/MS (MethodB) showed MH⁺=571; T_(RET)=2.87 min

Intermediate 1 (0.76 g, 1.33 mmol) was dissolved in ethanol (24 ml) andsubjected to catalytic hydrogenation over Lindlar Catalyst (0.095 g) for16 h. Catalyst was removed by filtration and evaporation of solvent invacuo gave Intermediate 2 (0.72 g, 99% yield) as a cream coloured foam.LC/MS (Method A) showed MH⁺=545; T_(RET)=2.24 min

Intermediate 2 (0.72 g, 1.32 mmol) was dissolved in tetrahydrofuran (7ml) and treated with N,N′-bis-(tert-butoxycarbonyl)-1-guanylpyrazole(0.45 g, 1.45 mmol). The mixture was stirred under a nitrogen atmospherefor 16 h. Volatiles were removed in vacuo to give a solid residue whichwas purified by Biotage flash chromatography; eluant initially EtOAc:Cyclohexane (1:1) then EtOAc: Cyclohexane (5:3). Evaporation of solventin vacuo afforded Intermediate 3 (0.48 g, 46% yield) as a white solid.LC/MS (Method A) showed MH⁺=787; T_(RET)=3.64 min

Intermediate 3 (0.48 g, 0.61 mmol) was dissolved in dichloromethane (19ml). The solution was cooled in an ice bath and trifluoroacetic acid (19ml) was added portionwise over 5 minutes. The mixture was then stirredfor 1 h under a nitrogen atmosphere before being allowed to warm toambient temperature and stirred a further 16 h. Volatiles were removedin vacuo, and the residue azeotroped from toluene to remove remainingtrifluoroacetic acid. Trituration with diethyl ether (20 ml) afforded awhite solid which was separated to give Intermediate 4 (0.50 g). LC/MS(Method B) showed (M−H)⁻=485; T_(RET)=0.52 min.

(4S, 5R,6R)-5-Acetylamino-4-azido-6-[(S)-4-nitrophenoxycarbonyloxy)-(2-oxo-[1,3]dioxolan-4R-yl)-methyl]-5,6-dihydro-4H-pyran-2-carboxylicacid methylester (see Eur. J. Med. Chem. 1999, 34, 563–574) (4.0 g) wasazeotroped with toluene (50 mL) and dissolved in MeCN (40 mL) andtriethylamine (1.12 mL) and 3-aminopropionic acid t-butyl esterhydrochloride (1.396 g) added. After 3 days at RT, the solvent wasremoved and the residue diluted with EtOAc (150 mL). This was washedwith 5% citric acid solution (2×50 mL), dried (MgSO₄) and concentrated.Purification by Biotage eluting with 1:1 cyclohexane: EtOAc, then 60:40then 65:35 cyclohexane: EtOAc gave Intermediate 6 as a colourless foam(3.45 g).

¹H-NMR (400 MHz, CDCl₃) δ 6.72 (d, 1H), 5.97 (d, 1H), 5.53 (t, 1H), 5.40(t, 1H), 5.03–4.95 (m, 2H), 4.92 (dd, 1H), 4.74–4.64 (m, 2H), 3.83 (s,3H), 3.64–3.54 (m, 1H), 3.38–3.27 (m, 2H), 2.65–2.56 (m, 1H), 2.52–2.43(m, 1H), 2.06 (s, 3H), 1.70 (s, 1H), 1.48 (s, 9H).

Similarly prepared to Intermediate 2 from Intermediate 6.

LC/MS (green method) MH⁺504, T_(RET)=2.22 min

Intermediate 8 was prepared similarly to Intermediate 3 fromIntermediate 7

LC/MS (green method) MH⁺744, T_(RET)=3.66 min

Intermdiate 8 (1.44 g), trifluoroacetic acid (20 mL), dichloromethane(20 mL) and anisole (2 mL) were stirred at RT for 3 h after which thevolatiles were removed in vacuo. The residue was triturated with Et₂O(2×25 mL) and then dried in vacuo to afford Intermediate 9 as a whitesolid (1.22 g).

LC/MS (green method) MH⁺488, T_(RET)=1.25 min

EXAMPLE 1 Preparation of Compound 9 by Reaction of Intermediate 5 andSuccinic Acid

The aminopropyl Intermediate 5 was prepared following a similar sequenceof steps to that described for the analogous aminobutyl Intermediate 4.

Succinic acid (4.21 mg, 0.0357 mmole), Intermediate 5 (50 mg, 0.071mmole) and benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP) (37.7 mg, 0.0852 mmole) were dissolved in DMF(2 ml) to which was added di-isopropylethylamine (DIPEA, 91.8 mg, 0.71mmole). The resulting mixture was stirred at room temperature for 5hours. The reaction mixture was purified by reverse phase HPLC using aWaters Symmetry C18 column (5 micron 19×100 mm), and gradient elution asshown in the following Table, to afford the protected dimer (12.8 mg,35%), MS 1027.4 (M+H)⁺

Time Flow rate (minutes) A % B % (ml/min) 0 100 0 6 2 100 0 6 22 40 60 632 40 60 6 35 100 0 6 42 100 0 6

-   A=Water containing 0.1% trifluoroacetic acid-   B=Acetonitrile containing 0.06% trifluoroacetic acid

The protected dimeric compound (12.5 mg, 0.0121 mmole) was dissolved ina mixture of water/methanol/triethylamine in the ratio 4:4:1 (1.5 ml)and stirred at room temperature for 1 hour then evaporated to drynessunder reduced pressure. Remaining triethylamine was removed by repeatedaddition of water and evaporation under reduced pressure. The remainingresidue was purified by reverse phase HPLC using a Waters Symmetry C18column (5 micron, 19×100 mm), and gradient elution as shown in thefollowing Table, to afford the dimer 9 as a white solid (7.5 mg, 65%)after freeze-drying.

Time Flow rate (minutes) A % B % (ml/min) 0 100 0 6 2 100 0 6 22 60 40 632 60 40 6 35 100 0 6 42 100 0 6 A = Water containing 0.1%trifluoroacetic acid B = Acetonitrile containing 0.06% trifluoroaceticacid

MS 474.4 (M+2H)²⁺, 946.8 (M+H)⁺

¹H-nmr (D₂O) δ (ppm): 1.64 (br, 4H); 1.94 (s, 6H); 3.06 (br, 4H); 3.17(m, 6H); 3.48 (dd, 2H); 3.63 (dd, 2H); 4.05 (m, 2H); 4.10 (dd, 2H); 4.39(dd, 2H); 4.49 (dd, 2H); 4.91 (dd, 2H); 5.82 (d, 2H).

EXAMPLE 2

Compounds Numbered 1–8, 10–14 and 21–25 in Table 1 were each prepared bycoupling the correct aminoalkyl compound (eg Intermediate 4 or 5) withthe appropriate dicarboxylic acid following similar conditions to thosedescribed in Example 1.

EXAMPLE 3

Compounds Numbered 15–18 in Table 1 were prepared from the carboxyIntermediate 9 by coupling with the appropriate diamine and thendeprotection following similar conditions to that given in Example 1.Compounds Numbered 19 and 20 in Table 1 were prepared from coupling ofthe glycine carboxy analog of Intermediate 9 with the appropriatediamine.

EXAMPLE 4

Evaluation of the Compounds of Formula (I)—Inhibition of Influenza VirusReplication

Cytopathic effect (CPE) assays were performed essentially as describedby Watanabe et al. (J. Virological Methods, 1994 48 257). MDCK cellswere infected with a defined inoculum of virus (determined byexperimentation to be the minimum sufficient to cause adequate CPE in 72hours and to be susceptible to control compounds at concentrationsconsidered to be consistent with published norms) in the presence serialdilutions of Compounds of the invention. Cultures were incubated for upto 72 hours at 37° C. in a 5% CO₂ atmosphere. The extent of CPE andhence viral replication was determined via metabolism of the viral dye3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)according to published methods (see for example, Watanabe et al., 1994).The compound concentration that inhibited CPE by 50% (ID₅₀) wascalculated using a computer program for curve fitting. InfluenzaA/Sydney/5/97 and B/Haribin/7/95 viruses were assayed and the resultsare shown in Table 1. Comparable data for a specifically disclosedcompound in WO 00/55149 and for compound A is also shown in Table 1.

TABLE 1 ID₅₀ μg/ml ID₅₀ μg/ml Description A/Sydney/5/97+ B/Harbin/7/95Compound A 0.023 +/− 0.024 0.013 +/− 0.011 Compound 1 0.0007 0.009Compound 2 0.0003 0.003 Compound 3 0.0003 0.002 Compound 4 0.0003 0.001Compound 5 0.0005 0.0009 Compound 6 0.0008 0.005 Compound 7 0.0003 0.001Compound 8 0.0004 0.001 Compound 9 0.0003 0.0003 Compound 10 0.00060.0007 Compound 11 0.0003 0.0003 Compound 12 0.0005 0.0002 Compound 130.0003 0.0002 Compound 14 0.0003 0.0002 Compound 15 >0.1 0.0002 Compound16 0.0005 0.0003 Compound 17 >0.1 0.0004 Compound 18 0.0003 0.0003Compound 19 >0.1 0.0002 Compound 20 >0.1 0.00005 Compound 21 0.00020.0003 Compound 22 0.0002 0.0003 Compound 23 0.0002 0.0003 Compound 240.0002 0.0007 Compound 25 0.0001 0.0003 Compound Number 8* 0.0007,0.0005 0.007 +/− 0.01  Compound Number 10* 0.057 >0.1 *As referenced inWO 00/55149

-   + Data provided in WO 00/55149 related to the virus H3N2 isolate    A/Victoria/3/75 rather than A H3N2 isolate A/Sydney/5/97. When    comparing such data the person skilled in the art will appreciate    that differences in antiviral potency are not uncommon for a given    compound when analysed against several different viruses in vitro.    For example, Woods et al (Antimicrob Agents Chemother 1993    37:1473–9) have reported that Compound A exhibits a wide range of    EC50 values (from 0.02 to 0.16 uM) in in vitro assays involving    recent clinical isolates. Accordingly, compound 8 was found to be    more potent in CPE assays involving the recent influenza A H3N2    isolate. A/Sydney/5/97 than the earlier H3N2 isolate    A/Victoria/3/75.    Data provided in Table 1 demonstrate that the compounds 1–25, in    addition to being substantially more potent than the highly active    compound A, are even more potent against A/Sydney/5/97 and    substantially more potent against the recent influenza B isolate    B/Harbin/7/95 than compounds 8 and 10 of WO 00/55149.

EXAMPLE 5

Plaque Reduction Assay

Madin Darby Canine Kidney (MDCK) cells are seeded into six well tissueculture plates and grown to confluency via standard methods. Influenzaviruses are diluted in a minimal volume of phosphate buffered salinesupplemented with 0.2% bovine serum albumin to yield an estimated titreof 50–100 plaque forming units (pfu) per well. After adsorption to theMDCK cells for one hour at 37° C. in a 5% CO₂ atmosphere the viralinocula is aspirated and replaced with viral growth media (minimalEagle's media supplemented with BSA, trypsin andinsulin/transferrin/selenium at optimal concentrations) containingsufficient agar or agarose (generally. 1–2%) to cause the media to gelat room temperature and at 37° C. in a 5% CO₂atmosphere until plaquesdevelop (generally 2–4 days). Plaques can be visualised with a suitablestain (e.g. 0.4% crystal violet in formal saline) before counting.Antiviral potency is expressed as the concentration of test articlewhich reduces plaque numbers by 50% of the untreated control value(EC₅₀).

EC₅₀ ng/ml PRA Example A/WSN* A/Vic* A/Syd* A/New* A/Pan* Compound A56, >100 5.5 +/− 8.2 2.4 0.27, 2.7, 3 0.23 1 0.05 <0.01, 0.02, 0.03, >10.006, 0.001 0.13 0.08, 0.21 Amantadine 220 11 157 Oseltamivir 0.11 0.230.3 *A/WSN/33 BVLV09 (H1N1) A/Victoria/3/75 BVLV017 (H3N2) A/Sydney/5/97BVLV015 (H3N2) A/New Caledonia/20/99 BVLV008 (H1N1) A/Panama/2007/99BVLV008 (H3N2) A/Bayern/7/95 BVL006 (H1N1) EC₅₀ ng/ml PRA Example B/Vic*B/Harb* B/HongK* B/Yam* Compound A 3, 20 0.19 21 +/− 6 0.2, 3.1 1 <0.01,0.1 0.014 0.316 0.032 Amantadine >10000 2061 Oseltamivir 32 0.7*B/Victoria/1/67 B/Hong Kong/5/72 BVLV012 B/Harbin/7/95 BVLV008B/Yamanashi/166/98 BVLV007

EXAMPLE 6

Assessment of Long Duration of Action

Rodents are anaesthetised and dosed with compound of interest by theintra-tracheal route at a dose volume of 0.8 ml/kg. The rodent is thenheld in the vertical position until full recovery is achieved. Atdifferent time points, for example, 2, 8, 24 and 48 hours post-dose,levels of compound in the lung tissue are assessed by analyticalmethods. Any analytical method suitable for detection of this type ofcompound may be used. The time at which levels of compound fall belowthe sensitivity of the analytical techniques identified will determinethe residency time of the compound in lung tissue.

The rat lung retention data for selected compounds is shown below.Please note that all experiments included a co-dosed internal standard,namely compound 3 of International Patent Publication No. WO 02/20514,to permit comparison. The data are expressed as a ratio with respect tothis compound, the structure of which is shown below.

The data for compound A is included for comparison purposes. Thecompounds of the invention have significantly greater retention at 7days than Compound A when expressed as a ratio of compound concentrationto standard concentration.

time Mean (PCT AU01/01128 Mean (PCT AU01/01128 Ratio Mean [lung] pointdose [cmpd] [cmpd] Compound 3) Compound 3) [cmpd]/PCT AU01/01128 hrsCompound mg/kg ng/g ng/g ng/g ng/g compound 3 48 Compound 18 0.1 565 442607 608 0.73 48 Compound 18 0.1 294 363 48 Compound 18 0.1 467 855 168Compound 18 0.1 150 161 351 349 0.46 168 Compound 18 0.1 98 243 168Compound 18 0.1 234 451 48 Compound A 0.1 421 352 698 1368 0.26(zanamivir) 48 Compound A 0.1 369 1901 (zanamivir) 48 Compound A 0.1 2671507 (zanamivir) 168 Compound A 0.1 91 61 815 750 0.08 (zanamivir) 168Compound A 0.1 47 925 (zanamivir) 168 Compound A 0.1 45 512 (zanamivir)

EXAMPLE 7

Alternative Assessment of Long Duration of Action and Efficacy

The protocol for infecting mice has been described previously (1–4).Mildly anaesthetised mice are inoculated into the external nares withinfluenza virus. Treatment procedure and regimen. A single dose ofcompound is administered at a defined time point up to 10 days prior toinfection, preferably 4–7 days prior to infection, or followinginfection, preferably immediately following infection and up to 48 hourspost infection. In most experiments, a non-lethal strain of influenza isused, and efficacy is assessed by reductions in lung virus titre. Formice given compound prior to infection, lungs are removed post infectioneither on a single day, or on days following infection, preferably days1–4 post infection. Homogenised lung samples are assayed for virus usingestablished methods, and the titres of viral load estimated and comparedto titres of virus in lungs of untreated mice.

In those experiments where a mouse-adapted lethal strain of influenza isused, efficacy is assessed by an increase in survival rate and/ornumbers of survivors, as compared to untreated mice.

REFERENCES

-   1. Ryan, D. M., J. Ticehurst, M. H. Dempsey, and C. R. Penn, 1994.    Inhibition of influenza virus replication in mice by GG167    (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is    consistent with extracellular activity of viral neuraminidase    (sialidase). Antimicrob. Agents and Chemother. 38 (10):2270–2275.-   2. von Itzstein M., W.-Y. Wu, G. B. Kok, M. S. Pegg, J. C.    Dyason, B. Jin, T. V. Phan, M. L. Smythe, H. F. White, S. W.    Oliver, P. M. Colman, J. N. Varghese, D. M. Ryan, J. M. Woods, R. C.    Bethell, V. J. Hogham, J. M. Cameron, and C. R. Penn. 1993. Rational    design of potent sialidase-based inhibitors of influenza virus    replication. Nature (London) 363:418–423.-   3. Woods, J. M., R. C. Bethell, J. A. V. Coates, N. Healey, S. A.    Hiscox, B. A. Pearson, D. M. Ryan, J. Ticehurst, J. Tilling, S. A.    Walcott, and C. R. Penn. 1993.    4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid is a    highly effective inhibitor both of the sialidase (neuraminidase) and    of growth of a wide range of influenza A and B viruses in vitro.    Antimicrob. Agents Chemother. 37:1473–1479.-   4. Robert J Fenton, Peter J Morley, Ian J Owens, David Gower, Simon    Parry, Lee Crossman and Tony Wong (1999). Chemoprophylaxis of    influenza A virus infections, with single doses of zanamivir,    demonstrates that zanamivir is cleared slowly from the respiratory    tract. Antimicrob. Agents and Chemother. 43, 11, 2642–2647.

1. A compound of general formula (I):

in which R is an amino or guanidino group; R² is acetyl ortrifluoroacetyl; X is CONH, NHCO or O; n is an integer from 2 to 6; andY is C₂–C₈ alkyl, C₃–C₈ cycloalkyl, C₁–C₄ alkoxyalkyl, an amino acid ordipeptide, or a pharmaceutically acceptable salt, ether, ester or saltof such ester thereof, with the proviso that when R is a guanidinogroup, R² is acetyl, X is O and n is 2, then Y is not C₂ alkyl.
 2. Acompound according to claim 1, in which R is a guanidino group.
 3. Acompound according to claim 1, in which R² is an acetyl group.
 4. Acompound according to claim 1, which contains a pharmaceuticallyacceptable salt, ether, ester or salt of such ester at one or more ofthe carboxyl groups, hydroxyl groups, amino groups or guanidine groups.5. A compound according to claim 1, in which said compound is an alkylester, an aryl ester or an acetyl ester.
 6. A method for the preparationof the compound of formula (I) according to claim 1 to 5 when X is CONHor NHCO, which comprises the steps of (a) coupling a compound of formula(II)

in which R² and n are as defined in claim 1, P₁ is a carboxylic acidprotecting group and X₁ is NH₂ or CO₂H; with a compound of formula (III)X₂—Y—X₂  (III) in which Y is as defined in claim 1 and X₂ is CO₂H or HN₂provided that it is not the same as X₁ to form a compound of formula(IV)

(b) deprotecting the compound of formula (IV).
 7. A pharmaceuticalformulation comprising a therapeutically effective amount of a compoundof formula (I) as defined in claim 1 or a pharmaceutically acceptablesalt, ether, ester or salt of such ester thereof, together with one ormore pharmaceutically acceptable carriers.
 8. A pharmaceuticalformulation according to claim 7, which comprises one or more anti-viralagents used to treat respiratory infections.
 9. A pharmaceuticalformulation according to claim 8, in which the agent is zanamivir,oseltamivir, amantadine, rimantadine, and/or ribavirin.
 10. An inhalerwhich comprises a compound according to claim
 1. 11. An inhaleraccording to claim 10 which is adapted for oral administration as afree-flow powder.
 12. An inhaler according to claim 10 which is ametered dose aerosol inhaler.
 13. A method for the treatment of anorthomyxovirus or paramyxovirus infection comprising the step ofadministration to a subject in need thereof of an effective amount of acompound of formula (I) as defined in claim
 1. 14. A method according toclaim 13 in which an orthomyxovirus or paramyxovirus infection is aninfluenza A or B infection, parainfluenza, mumps or Newcastle disease.15. A method according to claim 13 in which the administration is to therespiratory tract by inhalation, insufflation or intranasally or acombination thereof.